PROJECT SUMMARY: The growing incidences of antibiotic resistance is epitomized by the emergence of six multi-drug resistant bacteria referred to as the ?ESKAPE? pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. Without research to develop new antibiotics that are effective against these drug resistant pathogens, we risk regressing to a pre-antibiotic era where infections were a leading cause of morbidity and mortality. To counter this threat, the long-term goal of this research is to develop new antibiotics that function by targeting bacterial GroEL chaperonin systems. GroEL is a centralized molecular machine that maintains the proper structure and function of hundreds of other proteins. Thus, targeting this one molecular machine will have the cascading effect of inhibiting hundreds of proteins at once, the functional losses of which bacteria will not be able to recover from. A caveat to this strategy is that human cells have a mitochondrial counterpart, called HSP60, and there remains the possibility of inhibitor cross-talk that could lead to toxic effects on host tissues. The central hypothesis is that the structural and functional divergence between bacterial and mammalian chaperonins, as well as other mammalian proteins, will allow the selective targeting of small molecule inhibitors for GroEL and bacteria without toxic side effects to human cells. This hypothesis has been formulated on the basis of well-established findings on chaperonin structure and function presented in the literature, and preliminary data for chaperonin inhibitors produced in the applicants' laboratories. In particular, human HSP60 functions as a single-ring oligomer, which removes many of the allosteric transitions that regulate the function of double-ring bacterial GroEL. In addition, preliminary data suggest there are multiple distinct binding sites on bacterial GroEL that compounds can interact with to block chaperonin function. Thus, the possibility of small molecules targeting these unique binding sites to selectively inhibit bacterial GroEL over HSP60 and other human proteins is high. The overall objective of this proposal is to identify these unique allosteric binding sites, elucidate the structural/functional mechanisms of action for molecules binding to these sites, and define the selectivity profiles for site-specific inhibitors in vitro and in cells. The rationale for the proposed studies is that delineating the precise structural/functional mechanisms of action of chaperonin inhibitors will permit the rational development GroEL-targeting antibiotic candidates. The approach is innovative because it proposes a unique and unexplored strategy of exploiting proteostasis machinery ? specifically GroEL chaperonins ? for killing infectious bacteria. Without a fundamental shift in strategy for treating infectious diseases, antibiotic resistance will continue to rise and the prognosis for surviving infection by these superbugs will continue to worsen. Therefore, the proposed studies will have significant impact because the research findings will open novel therapeutic strategies for a broad range of intractable infectious diseases.